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Journal of Food Protection, Vol. 67, No.

12, 2004, Pages 000000


Copyright Q, International Association for Food Protection

Cloning of the Bile Salt Hydrolase (bsh) Gene from


Enterococcus faecium FAIR-E 345 and Chromosomal Location
of bsh Genes in Food Enterococci
AGUS WIJAYA, ANETTE HERMANN, HIKMATE ABRIOUEL, INGRID SPECHT, NUHA M. K. YOUSIF,
WILHELM H. HOLZAPFEL, AND CHARLES M. A. P. FRANZ*
Federal Research Centre for Nutrition and Foods, Institute of Hygiene and Toxicology, Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany
MS 04-132: Received 30 March 2004/Accepted 30 July 2004

ABSTRACT
?1

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in
a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene of a food E. faecium FAIRE 345 strain was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with
pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA
of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to
detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains
that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting
that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location
of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.

Bile acids are synthesized de novo from cholesterol


and are conjugated in the liver to either taurine or glycine
via an amide bond at the carboxyl C-24 position (21). The
conjugates are secreted into the intestine, where the amino
acids may be released from the conjugated bile acid by
bacterial conjugated bile salt hydrolases (Bsh) (6, 15, 21).
Bsh activity has been described for members of several bacterial genera, including Listeria, Bifidobacterium, Clostridium, Bacteroides, Lactobacillus, and Enterococcus (13, 9,
10).
The lowering of blood cholesterol concentrations in
hypercholesterolemic humans as a result of Bsh activity of
probiotic bacteria is one of the suggested functional effects
of probiotics (13). However, this possible effect is controversial with regard to its validity and the mechanisms involved (14, 24). In contrast to this desirable or probiotic
effect, Bsh activity may also have adverse effects, in that
extensive deconjugation of bile salts in the human small
bowel can lead to steatorrhea and an increased formation
of secondary (dehydroxylated) bile salts, which are cytotoxic and cocarcinogenic (14). The ecological significance
of bacterial Bsh activity has also been subject to debate.
De Smet et al. (3) suggested that Bsh activity serves as a
detoxification reaction for lactobacilli. However, Moser and
Savage (15) tested a wide spectrum of Bsh1 lactobacilli for
their resistance to bile salt toxicity and could not find evidence to support this hypothesis. They then suggested (15)
that instead of protecting lactobacilli from toxicity of con* Author for correspondence. Tel: 149 721 6625 225; Fax: 149 721 6625
453; E-mail: charles.franz@bfe.uni-karlsruhe.de.

jugated bile salts, Bsh activity may rather be important for


bacterial colonization of and growth in the intestine. Such
abilities may be beneficial in the case of probiotic strains,
for which colonizing ability is viewed as a positive characteristic (13), but may be viewed as detrimental in the case
of pathogenic bacterial strains, for which colonization ability is regarded as a virulence factor (7). For Listeria monocytogenes, Bsh activity is a regulated virulence factor involved in colonization of the host during the intestinal and
hepatic phases of listeriosis (4).
The bsh genes from gram-positive bacteria such as L.
monocytogenes (10), Lactobacillus plantarum (1), Lactobacillus gasseri (19), Lactobacillus johnsonii (6), Lactobacillus acidophilus (GenBank accession no. AF091248.3),
Bifidobacterium longum (24), and Clostridium perfringens
(2) have been cloned and characterized in detail. In addition, the bsh genes of some enterococci have also been
cloned and sequenced (16, 22).
Enterococci are an important group of lactic acid bacteria. Some strains are used as probiotics, and the enterococci also regularly occur in numerous fermented foods,
including sausages and cheeses (7). Bsh activity is quite
common among strains of enterococci from foods (9). In
recent years, strains of enterococci have also become acknowledged as major human pathogens involved in nosocomial infections. These pathogenic strains express a variety of virulence factors related to colonization and cytotoxicity (12). Enterococci from food can harbor virulence factors (5, 8), and there are concerns that enterococcal strains
from food can transfer genes encoding virulence factors or
antibiotic resistance to human intestinal strains. This study

WIJAYA ET AL.

J. Food Prot., Vol. 67, No. 12

TABLE 1. Enterococcus strains and plasmids used in this study


Enterococcus strain or plasmid

FAIR-E straina
E. durans: E 326, E 332
E. faecium: E 3, E 6, E 14, E 15, E 20, E 25, E 34, E 84,
E 154, E 170, E 225, E 227, E 243, E 263, E 345, E 349
E. faecalis: E 69, E 71, E 237, E 313
E. faecalis FAIR-E 29
E. faecium FAIR-E 80
E. faecalis MMH594b
E. faecalis MMHV583b
E. faecalis FAIR-E 404
E. faecalis FAIR-E 313
E. faecalis FAIR-E 77
Plasmid
pAW01

Relevant characteristic, source

Bsh1

Bsh2
Esp1, control
Ace1, control
AS1, control
Gel1, control
Cyl1, control
pSKII 6 containing a 3.3-kb chromosomal DNA fragment from
E. faecium FAIR-E 345
pUC19 containing 830-bp fragment of the bsh gene from
E. faecium FAIR-E 345

pAW02
a

FAIR-E, research collection of the EU-project FAIR-CT-3078 deposited in the BCCM/LMG Bacteria Collection Laboratorium voor
Microbiologie, University of Gent, Gent, Belgium.
b Strains kindly donated by V. Shankar.

describes the cloning of a bsh gene from Enterococcus faecium strain FAIR-E 345 isolated from food and the determination of the genomic location of bsh genes among Bsh1
enterococci to assess the likelihood of transfer of the Bsh
activity trait by conjugation.
MATERIALS AND METHODS

?3

Bacterial cultures and growth conditions. All enterococci


were grown aerobically in MRS (de Man Rogosa Sharpe) broth
(Merck, Darmstadt, Germany) at 378C. Bsh1 enterococci used in
this study were isolated from food (9) and comprised Enterococcus faecalis, E. faecium, and Enterococcus durans strains (Table
1). Stock cultures were maintained at 2808C in MRS broth with
15% glycerol.
Detection and quantification of Bsh activity. In a previous
study (9), Bsh activity was detected on MRS agar plates supplemented with 0.5% (wt/vol) sodium salt of taurodeoxycholic acid
(Sigma, St. Louis, Mo.) and 0.37 g/liter CaCl2. The size of the
precipitation zone on the screening medium was not correlated
with the actual BSH activity as measured by high-pressure liquid
chromatography (HPLC) (9). Because the plate screening assay
also sometimes does not result in large zones of precipitates
around colonies and is therefore difficult to interpret for certain
strains (9), the Bsh activity of E. faecium FAIR-E 345 was also
quantified by HPLC using methods previously established (9),
which involved measuring the deconjugation during 6 h of incubation of glycocholic acid (GCA; G-2878, Sigma) added at an
initial concentration of approximately 2 mg/ml to the E. faecium
FAIR-E 345 culture. A standard stock solution of GCA (100 mg/
ml) was used as a reference for HPLC to calculate the amount of
GCA deconjugation during incubation of the culture as described
previously (9). Deconjugation of GCA was measured by HPLC
in three independent experiments.
Detection of virulence factors among Bsh1 enterococci.
The presence of virulence factors was tested for E. faecalis and
E. durans strains in this study, whereas the presence of virulence

traits for E. faecium strains were determined previously (8). Genes


for cytolysin (Cyl), enterococcal surface protein (Esp), gelatinase
(Gel), and aggregation substance (AS) were amplified using the
primers and methods described by Franz et al. (8) or Eaton and
Gasson (5). PCR amplification was also used to detect the gene
for the adhesin to collagen (Ace) using primers Ace-1 (59-GAA
TTG AGC AAA AGT TCA ATC G-39) and Ace-2 (59-GTC TGT
CTT TTC ACT TGT TTC-39). For PCR of the ace gene, DNA
was amplified in a 50-ml volume containing 100 ng of template
DNA, 200 mM concentrations of dNTPs, 50 pM of the respective
primers, 1.5 mM MgCl2, 2 U of Taq DNA polymerase (Amersham Pharmacia, Freiburg, Germany), and 13 reaction buffer
(Amersham Pharmacia). DNA was amplified in 35 cycles (denaturation at 948C for 1 min; annealing at 548C for 1 min; extension
at 728C for 1.5 min). Gelatinase activity was also tested on ToddHewitt agar (Difco, Becton Dickinson, Sparks, Md.), as described
previously (8).
Cloning of the bsh gene. Genomic DNA from E. faecium
FAIR-E 345 was isolated according to the methods of Quadri et
al. (18). Genomic DNA was partially digested with Sau3AI (New
England Biolabs, Frankfurt am Main, Germany) and subjected to
electrophoresis on a 1.5% agarose gel. Following electrophoresis,
fragments of various sizes (1.5 to 2.5 kbp, 1.5 to 3.0 kbp, 2.5 to
4.0 kbp, 2.5 to 4.5 kbp, and 4.5 to 6.0 kbp) were extracted directly
from the agarose gel using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Vector pSKII1/2 was digested with
BamHI and dephosphorylated using calf intestinal phosphatase
(New England Biolabs). The vector was ligated to the DNA fragments, and constructs were used for electrotransformation into
electrocompetent Escherichia coli TOP10 cells (Invitrogen, Groningen, the Netherlands). Transformed cells were spread plated
onto Luria Bertani (LB) agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and isopropyl-beta-D-thiogalactopyranoside at standard concentrations (20) and 150 mg/ml ampicillin.
Plates were incubated at 378C overnight. Colonies containing an
insert were selected, and replicate were plated onto LB agar and
LB-based differential medium as described by Christiaens et al.

J. Food Prot., Vol. 67, No. 12

CHROMOSOMAL LOCATION OF FOOD ENTEROCOCCI

BSH

GENES

(1) to detect Bsh-positive clones. The plates were incubated anaerobically at 378C for 2 days, and the presence of a white precipitation zone around the colony on differential medium was indicative of Bsh activity (1).
DNA sequencing and homology searches. Plasmid pAW01
isolated from a Bsh-positive clone was isolated using a plasmid
midi kit (Qiagen), and the DNA was sequenced bidirectionally
using M13 universal and custom-made primers in a primer walking strategy. Sequencing was done at GATC (Konstanz Biotech,
Germany). The DNA sequence was analyzed using the DNA
STAR program and submitted to GenBank (accession no.
AY260046). A GenBank database search was done to check homology of the amino acid sequence against known protein sequences. Homology among bile salt hydrolases was determined
using the DNASTAR program and ClustalW alignment of amino
acid sequences contained in the GenBank databank.
Preparation of a gene probe and determination of the
genomic location of bsh genes. The bsh gene was amplified by
PCR using primers Aw-01 (59-TAT ATC TAG AGG AGT AAT
ATG AAC GTG GA-39) and primer Aw-02 (59-TAT AGC ATG
CAA ATA ATC AAT CAC AAC ACA C-39) complementary to
the 59 and 39 ends of this gene in pAW01, respectively, and containing XbaI and SphI restriction enzyme sites (underlined). DNA
was amplified in a 50-ml volume, and the PCR mixture contained
100 ng of template DNA, 200 mM concentrations of dNTPs, 25
pM of each of the respective primers, 1 U Taq polymerase (Amersham Pharmacia), and 13 reaction buffer (Amersham Pharmacia).
DNA amplification was done in 32 cycles (denaturation at 948C
for 1 min; annealing at 528C for 1 min; extension at 728C for 1
min). The resulting PCR fragment was cloned into the XbaI and
SphI sites of plasmid pUC19, resulting in plasmid pAW02. Plasmid pAW02 was digested with the restriction enzyme XbaI to
linearize the plasmid and was labeled with digoxigenin using the
DIG dUTP labeling and detection kit (Roche, Mannheim, Germany) according to the manufacturers instructions. Plasmid DNA
was isolated from Bsh-positive enterococci (7) according to the
method of van Belkum and Stiles (25), and total genomic DNA
was isolated using the method of Pitcher et al. (17). Total genomic
DNA was also isolated from two Bsh2 enterococcal strains (E.
faecium FAIR-E 80 and E. faecalis FAIR-E 29) and was used as
a negative control to confirm the specificity of the Bsh gene probe.
Total genomic DNA was digested with XbaI and HindIII and subjected to electrophoresis using a 1.4% agarose gel. Plasmid DNA
was subjected to electrophoresis on these agarose gels without
restriction enzyme cutting. DNA was transferred to a Hybond N1
membrane by Southern blotting according to standard techniques
(20), and after Southern transfer the agarose gel was stained with
ethidium bromide and visualized under UV light to check whether
DNA had successfully transferred. The DIG-dUTPlabeled probe
was used to detect the location of the bsh gene according to the
DIG dUTP manufacturers instructions. Prehybridization was carried out at 658C for 4 h, and hybridization was carried out at 688C
overnight.

RESULTS AND DISCUSSION


Detection and quantification of Bsh activity by E.
faecium FAIR-E 345. When E. faecium FAIR-E 345 was
spotted onto MRS agar plates supplemented with the sodium salt of taurodeoxycholic acid and CaCl2, a white zone
of precipitation indicative of Bsh activity was formed. Detection of GCA by HPLC revealed that the E. faecium
FAIR-E 345 strain deconjugated this compound in the me-

FIGURE 1. Numbers of E. faecium FAIR-E 345 and Bsh activity


as indicated by decreasing glycocholic acid (GCA) concentrations
during growth in MRS broth containing 2 mg/ml GCA for 6 h.
Results shown are mean values from three replicate experiments;
the standard error is indicated.

dium from an initial concentration of 2,000 mg/ml to approximately 300 mg/ml during the 6-h incubation period
(Fig. 1). During this period, no noticeable growth took
place; however, cell numbers did not decrease noticeably
(Fig. 1) and thus the cultures were considered to be metabolically active.
Nucleotide sequence and amino acid homology. One
of approximately 4,000 colonies screened after transformation of E. coli TOP10 with chromosomal DNA fragments of 2.5 to 4.0 kb expressed the bsh gene, as was demonstrated by the presence of a large zone of precipitation
around the colony growing on LB-based differential agar.
The insert in the plasmid pAW01 contained by this clone
was completely sequenced in both directions. Analysis of
the DNA sequence revealed the presence of one open reading frame (bsh) encoding a protein of 324 amino acids with
a pI of 4.877. A presumptive ribosome binding site
(GGAGGAA) was located eight bases upstream of the ATG
start codon for this open reading frame. Presumptive 210
(TATAGT) and 235 (TTGATA) promoter sequences were
located upstream of this ribosome binding site. In addition,
one indirect and one direct repeat sequence were noted upstream of the promoter region (GenBank accession no.
AY260046). A possible terminator with dyad symmetry
started 2 bp upstream of the TGA stop codon of this open
reading frame. A databank search indicated that the amino
acid sequence deduced from this open reading frame had
the highest homology with the identical Bsh protein sequences of E. faecalis strains V583 and MMH594 (82.7%
identity with both) and lower homology with the Bsh proteins from L. monocytogenes (77.8% identity), L. plantarum
(68.5% identity), L. johnsonii (52.8% identity), L. gasseri
(49.2% identity), C. perfringens (42.2% identity), B. longum (38.2% identity), and L. acidophilus (34.8% identity)
(1, 2, 6, 10, 19, 24). In addition, the Bsh protein from E.
faecium FAIR-E 345 also had some homology with the
penicillin V acylase (Pva) of Bacillus sphaericus (32.7%
identity) (23).
The amino acids of the active site of Pva were determined by crystal structure analysis (23) to be Cys-1, Asp20, Tyr-82, Asn-175, and Arg-228. As pointed out by Ta-

WIJAYA ET AL.

J. Food Prot., Vol. 67, No. 12

FIGURE 2. Alignment of the Bsh protein sequences from E. faecium FAIR-E 345 (BSH Efm), E. faecalis strains MMH594 and V583
(Efs), L. monocytogenes (BSH Lm), B. longum (BSH Bl), C. perfringens (BSH Cp), L. acidophilus (BSH La), L. gasseri (BSH Lg), L.
plantarum (BSH Lp), and L. johnsonii (BSH Lj) by ClustalV alignment. Identical amino acids are indicated in solid and shaded boxes;
amino acids hypothesized to be part of the active site are indicated in solid boxes.

naka et al. (24), all Bsh proteins characterized so far also


have these conserved amino acids at these positions, with
the exception of Tyr-82, which is replaced by Asn-81 in
the Bsh proteins. These positions also were conserved for
the amino acid sequence for the Bsh protein of E. faecium
FAIR-E 345, as deduced from the nucleotide sequence of

the bsh gene in this study (Fig. 2). Alignment of the Bsh
proteins from enterococci revealed that apart from these
conserved residues these molecules have generally high homology of amino acid sequences, and the greatest heterogeneity among amino acids was found at the carboxyl end
of the molecule (Fig. 2).

J. Food Prot., Vol. 67, No. 12

CHROMOSOMAL LOCATION OF FOOD ENTEROCOCCI

BSH

GENES

Tanaka et al. (24) purified the Bsh protein and determined that the N-terminal amino acid sequence for purified
Bsh did not include a Met residue. The authors thus determined that the formylmethionine was processed and that
Cys was actually the first amino acid of the mature protein.
This finding was also obtained with Pva, in which Cys-1
plays a central role at the active site (23, 24). Using sitedirected mutagenesis, Tanaka et al. (24) showed that when
Cys-1 was replaced with Ala Bsh activity was completely
abolished. The highly conserved Cys residue in all Bsh proteins (Fig. 2) confirms the importance of Cys at the active
site. This finding suggests that for all the Bsh proteins
shown aligned in Figure 2, the formylmethionine is processed and Cys is the first amino acid of the mature Bsh
protein. If this is the case, the Bsh protein from E. faecium
FAIR-E 345 would consist of 323 amino acids and the protein would have a pI of 4.877.
Probable genomic localization of the bsh gene
among Enterococcus strains. Use of the bsh probe produced clear hybridization signals when probing the genomic DNA of some of the strains (Fig. 3, a and b). When
the bsh gene probe was hybridized to the genomic DNA of
the negative control strains E. faecalis FAIR-E 29 and E.
faecium FAIR-E 80, only a very weak signal was observed
(Fig. 3, c and d) with DNA from the latter strain. This
signal was noticeably weaker and easily distinguished from
the strong positive signals obtained with most of the Bsh1
strains (Fig. 3). The probe hybridized and positive signals
were obtained with the genomic DNA of 22 enterococci
strains (Table 2). Although 10 of these strains harbored
plasmid DNA, the probe hybridized to only the total genomic DNA and not to the plasmid DNA of these strains
(Table 2). For 12 other enterococcal strains that contained
plasmids, weak signals were obtained only when probing
the total genomic DNA. These weak signals could represent
hybridization signals for the bsh genes but could not be
unequivocally differentiated from the negative control. This
finding suggests that there is a certain degree of heterogeneity among the nucleotide sequences of bsh genes of enterococci that did not allow the probe to bind equally well
in all cases, resulting in signals of the same intensity. Nevertheless, in this study hybridization signals could not be
detected from plasmid DNA preparations of any of the
strains tested. Therefore, our results indicate that the probe
could in many cases specifically detect the bsh gene and
that this gene is probably located on the chromosome in
enterococci strains. Such a chromosomal location of the bsh
gene would be consistent with all previous reports on the
cloning of these genes from E. faecalis, L. monocytogenes,
lactobacilli, B. longum, and C. perfringens (1, 2, 6, 10, 20,
25). However, the possibility that the bsh gene resides on
a conjugative transposon that is integrated into the chromosome cannot be ruled out.
For E. faecalis strains V583 and MMH594, the bsh
genes were located on the chromosome on so-called pathogenicity islands together with genes encoding virulence
factors such as Cyl, AS, and Esp (16, 22). Although such
pathogenicity islands are often thought to be mobile, filter-

FIGURE 3. Agarose gel with total genomic DNA from enterococcal strains. DNA was cut with the restriction enzymes XbaI and
HindIII (a and c), transferred to a nylon membrane after Southern
blotting of the agarose gels, and probing with the BSH probe (b
and d). (a and b) Lane 1, 1-kb DNA ladder; lane 2, Enterococcus
faecium FAIR-E 6; lane 3, E. faecium FAIR-E 14; lane 4, E.
faecium FAIR-E 15; lane 5, E. faecium FAIR-E 20; lane 6, E.
faecium FAIR-E 25; lane 7, E. faecium FAIR-E 34. (c and d)
Lanes 1 and 5, 1-kb marker; lane 2, negative control E. faecalis
FAIR-E 29; lane 3, positive control E. faecium FAIR-E 345; lane
4, negative control E. faecium FAIR-E 80. Arrow indicates position of weak band for signal obtained with negative control E.
faecium FAIR-E 80 (d).

mating transfer studies have revealed that the pathogenicity


island cannot be transferred to another E. faecalis recipient
strain, suggesting that transfer of the pathogenicity island
occurs on an evolutionary rather than a laboratory scale
(22). Our results indicate that most of the food enterococcal
isolates possess few if any virulence factors, with the exception of E. faecalis strains FAIR-E 69 and FAIR-E 71,
which possessed the genes for Ace, AS, and Esp and those
for Gel, Ace, and Esp, respectively (Table 3). Because of
this lack of virulence factors in most strains, a similar lo-

WIJAYA ET AL.

J. Food Prot., Vol. 67, No. 12

TABLE 2. Presence of plasmid DNA among Bsh-positive enterococci and hybridization of bsh gene probe to plasmid or total genomic
DNA preparations
Enterococcus
species

Strains with plasmid DNA

Hybridization signal of
plasmid DNA with bsh
gene probe

Strong hybridization of total


genomic DNA
with bsh gene probe

E 326, E 332
E 3, E 6, E 14, E 15, E 20, E 25, E 34,
E 84, E 154, E 170, E 225,
E 227, E 243, E 263, E 345, E 349
E 69, E 71, E 237, E 313

E. durans
E. faecium

2
E 3, E 6, E 20, E 25, E 84,
E 243

2
2

E. faecalis

E 69, E 71, E 237, E 313

calization for the bsh genes on such a pathogenicity island


and a similar organization of bsh genes and virulence genes
as seen in the E. faecalis strains MMH594 and V583 (16,
22) seems unlikely.
If Bsh activity is regarded as an important functional
property of probiotic enterococci strains, the presumptive
chromosomal location of Bsh genes would imply that this
trait is likely to be stable in Bsh1 probiotic enterococci and
thus is not likely to be influenced by plasmid loss. However,
if Bsh activity is considered a colonization factor favoring
intestinal growth, as suggested by Moser and Savage (15),
it could be viewed as a potential virulence factor, especially
in enterococcal strains that carry other recognized virulence
traits. Therefore, it is unlikely that this trait is transferable
by conjugative plasmids from Bsh1 food strains or probiotic strains that carry no virulence factors to other food
strains or commensals that do harbor virulence traits. However, such speculations are based on the assumption that
bsh genes in enterococci are not located on conjugative
transposons, and this possibility requires further investigation.
ACKNOWLEDGMENTS

REFERENCES
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This study was carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD program CT97-3078, Enterococci in Food Fermentations:
Functional and Safety Aspects. The opinions expressed here do not necessarily reflect the Commissions views and in no way anticipate the Commissions future policy in this area. A. Wijaya was supported by a scholarship from the German Academic Exchange Service, which is gratefully
acknowledged.

9.

10.

TABLE 3. Presence of the genes for the virulence factors gelatinase (Gel), cytolysin (Cyl), aggregation substance (AS), adhesin
to collagen from E. faecalis (Ace), or enterococcal surface protein
(Esp) in Bsh-positive enterococci from food
Presence of gene for virulence factor
Enterococcus strain

Gel

Cyl

AS

Ace

Esp
11.

E 3, E 6, E 14, E 15, E 20,


E 25, E 34, E 84, E 154
E 70, E 225, E 227,
E 243 E 263, E 345
E 349
E 69
E 71
E 313

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2
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2
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